We are investigating the role of nucleotides in regulation of liver growth. Evidence from our own and other laboratories has suggested possible participation of cyclic nucleotides, and possible involvement of certain unusual nucleotides as well. As cell cultures offer advantages over whole animal experimentation we have initially established a system for studying adult rat hepatic parenchymal cells cultured as monolayers, in a serum-free medium virtually free of contamination with other cell types. These cells respond to stimulation of 3H-thymidine incorporation into DNA by epidermal growth factor (EGF), insulin and glucagon; this observation confirms and extends the original observations of Richman et al., who employed mixed populations of liver-derived cells in the presence of serum (Proc. Natl. Acad. Sci. 73:3589, 1976). We find that replacement of glucagon in the EGF-insulin-glucagon mixture with dibutyryl cyclic-AMP or substances that increase the intracellular cyclic AMP concentration (e.g., cholera toxin, isoproterenol, methylisobutylxanthine) does not alter the experimental outcome. We will now examine the timing and extent of the cyclic-AMP rises, and cyclic-AMP turnover in relation to stimulation of DNA synthesis by these and other putative growth factors, and also explore the possible involvement of the other cyclic nucleotides, and various highly phosphorylated nucleotides as well.